Article Addendum

www.landesbioscience.com Bacteriophage e28265-1

Bacteriophage 4, e28265; January–december; © 2014 landes Bioscience

Article Addendum

The sequencing of marine metage-nomic fosmids led to the dis-

covery of several new complete phage genomes. Among the 21 major sequence groups, 10 totally novel groups of marine phages could be identified. Some of these represent the first phages infect-ing large marine prokaryotic phyla, such as the Verrucomicrobia and the recently described Ca. Actinomarinales. Coming from a single deep photic zone sample the diversity of phages found is astonishing, and the comparison with a metavirome from the same location indi-cates that only 2% of the real diversity was recovered. In addition to this large macro-diversity, rich micro-diversity was also found, affecting host-recognition modules, mirroring the variation of cell surface components in their host marine microbes.

Introduction

The problem of retrieving microbes in pure culture has hampered enor-mously the development of microbiol-ogy. For many years now, it has been clear that the microbes stored in culture collections only represent a tiny slice of the real diversity of microbes in nature.1 Furthermore, the recent development of comparative genomics has revealed that the gene diversity displayed by an iso-late is a very poor representation of the enormous genetic diversity contained in prokaryotic2 and apparently, although at much smaller scale, also in some pro-tist3 species, the so-called pan-genome. It seems clear now that a good representa-tion of the genomic diversity of microbes

cannot be acquired within a sensible time frame by the classical approach of cultur-ing and sequencing individual isolates. A new avenue was found by pioneers such as Edward DeLong and J Craig Venter that applied genomic shot gun sequencing to microbial communities providing infor-mation about their collective genomes, the metagenome.4,5 Later the develop-ments in high throughput, new genera-tion sequencing technologies have opened the field of metagenomics to most micro-biologists decreasing the cost and techni-cal difficulties.6,7 A major step forward to understand microbes in nature is in the making.

However, a fundamental gap was hith-erto left largely unfilled. If the cultiva-tion of cellular microbes is demanding, what of their accompanying non-cellular viral predators? In aquatic environments there are, as a rule of thumb 10 phages per cell.8,9 Even assuming that the typi-cal phage genome is ca. 100 times smaller than the one of the host the amount of genetic diversity that they carry is size-able.10 In addition, phages are an essential component of microbial communities that contribute to the evolution and diversity of the prey species.11 However, the develop-ment of a parallel description of the com-munity virome (metavirome) has been hampered by the relatively small amount of DNA available in the viral fraction. The use of genomic amplification based approaches (mostly multiple displacement amplification or MDA) bias heavily most available metaviromes.12 In addition, the annotation of phage genes, always chal-lenging, becomes extremely difficult using the small reads generated by the presently

Tales from a thousand and one phages

Francisco Rodriguez-Valera,* Carolina Megumi Mizuno, and Rohit Ghai1Evolutionary Genomics Group; Universidad Miguel Hernandez; Campus de San Juan; Alicante, Spain

Keywords: metagenomics, metavirome, marine phages, deep chlorophyll maximum, constant-diversity, red-queen, phage evolution, pan-selectome, pan-genome

Submitted: 01/14/2014

Revised: 02/17/2014

Accepted: 02/17/2014

Citation: Rodriguez-Valera F, Mizuno CM, Ghai R. Tales from a thousand and one phages. Bacteriophage 2014; 4:e28265; http://dx.doi.org/10.4161/bact.28265

*Correspondence to: Francisco Rodriguez-Valera; Email: frvalera@umh.es

Addendum to: Mizuno CM, Rodriguez-Valera F, Kimes NE, Ghai R. Expanding the marine virosphere using metagenomics. PLoS Genet 2013; 9:e1003987; PMID:24348267; http://dx.doi.org/10.1371/journal.pgen.1003987

e28265-2 Bacteriophage Volume 4

available high throughput sequencing techniques.13

Serendipitously, we have found an alternative to dig into the natural diversity of phages. Very early in the development of metagenomics the presence of typical viral genes in cellular metagenomes was described.14,15 Gradually it became evident that this was just the result of the retrieval of cells undergoing the lytic cycle16 in the cellular fraction. Many phages replicate forming long genome concatamers17 and when the DNA from the cells in which they are replicating is collected, large amounts of the phage genome becomes available, actually amplified in a natural way. We described some years ago a method to sequence fosmid clones by NGS.18 Since the insert in a fosmid clone is similar to the size of many phage genomes (30–40 Kb), fosmids provide a convenient way to generate natural contigs derived from these phage genomes amplified in the metagenomic DNA. We did a pilot study in which a set of 130 randomly selected clones were sequenced and 16 long contigs that clearly belonged to bacteriophages could be assembled.16 Further analyses suggested that these phage-derived contigs actually belonged to five different sets of highly related siphoviruses and they likely infected picocyanobacteria, particularly Synechococcus. Importantly, three of these represented complete genomes.

Given the success in this relatively straightforward approach to obtaining phage genomes, we sequenced a much larger set of fosmids (ca. 6000), which provided more than a thousand con-tigs, of which 208 were complete phage genomes from a single sample taken from the deep (50 m) chlorophyll maximum in the Western Mediterranean.19 This is the region of maximum cell density in the water column where infection rate is probably high. Actually, ca. 10–15% of fosmid sequences retrieved from the cel-lular fraction DNA corresponded to phage genomes.19

The Local Diversity

The 1148 phage contigs were orga-nized into increasingly larger groups using three different stages of clustering.19 In the first stage, contigs with > 95% nucleotide

identity and > 95% coverage were clus-tered to identify nearly identical sequences (the largest number of identical contigs was 22 and the mean 5). In the second, the coverage criterion was reduced (from > 95% to > 20%) to gather more con-tigs for each previous cluster. These two stages enabled creating groups of contigs that had very high nucleotide identity to each other and also likely originating from the same lineage of phage genome. Even though the majority of the contigs (913) could be clustered with a high nucleotide identity (95%) in 117 different clusters, 235 contigs were singletons and remained unclustered. The overlaps within these contigs that probably derive from phage replicating concatamers also enabled us to assess genome completeness, and 208 complete genomes could be identified. The third clustering stage was based on protein sequence similarities in order to identify much more distant relationships within the 208 complete phage genomes to reference phage genomes, what allowed us to define 10 novel yet undescribed groups of phages within this collection (Fig. 1).

The largest clusters could be associated with cyanophages (largest cluster with 102 contigs) and pelagiphages (largest cluster with 40 contigs) (also shown in Fig. 1). Such contigs are the most likely to belong to active phages that were replicating by concatamer formation when the sample was taken (though they may not be exactly the most abundant in the sample). Apart from phages infecting picocyanobacteria and the SAR11 group microbes, we also identified contigs that likely infect marine Verrucomicrobia20 (2 contigs, 1 complete genome) and low GC Actinobacteria21 (a single contig, incomplete genome), that represent the first phages from the marine habitat for these two groups.

The availability of large numbers of highly related contigs in these clusters allowed examining genuinely concurrent phage populations, something that with cultured isolates has only been possible to a limited extent.22,23 Broadly, aside from the identical fragments derived from a single concatamer, three different patterns in phage genome diversity were discern-ible (Fig. 2). The first was the recovery of phage genome fragments that were nearly

identical to each other along their entire lengths except for small single nucleotide polymorphisms and indels. Such highly related versions may represent the low-est levels in phage genome population diversity. These are possibly the closest approximations of a natural phage popu-lation to a clonally derived genome in pure culture. Next, we found genomes sharing long, nearly identical regions, but with significant sequence divergence in smaller regions, that although often preserving synteny had much lower or no similar-ity (Fig. 2). Typically these regions con-tained host-recognition genes (e.g., tail fiber) but also others (e.g., terminases and capsids). Though this might suggest that such divergence is the result of gradually evolving host-specificity, other expla-nations are possible (see below). These “genomic islands” actually have a clear parallel in prokaryotic genomes, although in them gene content seems to be more variable i.e., synteny is less preserved. An apparent feature that emerged from these comparisons, apart from the patterns dis-cussed above, was the mosaic nature of the genomic architecture in these concurrent populations (Fig. 2). Given the extremely high nucleotide identity (> 95%) in large parts of these contigs, the presence of such a hybrid architecture can only be explained by recombination during mixed infections of the common host.

In any case, it seems clear that even with the more than a thousand large genomic fragments described we were far from covering even a small fraction of the diversity of phages present in this single Mediterranean sample. Comparing the assembled phage contigs with a meta-virome from the same location we have calculated that barely 2% of the diversity found in the metavirome (15 Gb) was represented in the combined 26 Mb of sequences assembled in the fosmids.

The Same Phages Everywhere

A controversial issue in microbiol-ogy is the existence of a biogeography of microbes. Recent evidence from metage-nomics and genomics of marine bacteria indicates a weak geographic association at the strain level, although most spe-cies are global along the broad band of

www.landesbioscience.com Bacteriophage e28265-3

oligotrophic temperate and tropical ocean around the Earth.24,25 Regarding phages there seems to be evidence of widespread distribution, but based only on short over-laps.13 We have retrieved examples19 that indicate a global distribution of phages throughout these ocean water masses (altogether close to half the surface of the Earth). For example the recovery of a cyanophage genome fragment more than 40 Kb and 97% similarity (nucleotides) to an isolate obtained off the coast of Southern California (Fig. 2). Along the same lines, the finding of nearly identical versions of the hyper-diverse (see below) tail fiber protein (Fig. 2) in a pelagiphage from Bermuda26 and our Mediterranean sample all point toward very similar popu-lations found at vastly separated ocean sites. Of course the global ocean is a con-tinuum in which eventually all the water gets mixed, but it is an extremely slow process that takes in the range of thou-sands of years.27 Airborne transportation

in extreme meteorological events might provide faster connection among marine microbial communities that travel in the aerosols formed for example in large storms, but once they arrive to the distant places they would face a huge dilution compared with the local strains. It is pos-sible also that these populations are con-served under the similar conditions of the deep photic zone worldwide because they are selected for28 (see below).

Is the Red Queen Running or is it a Zoetrope?

The presence of simultaneous, closely related lineages displaying enormous microdiversity, either in prokaryotic or phage genomes gives rise to two different interpretations. One in which populations are viewed as fast-evolving assemblages where both, microbes and their phages, are in a constant arms-race (the Red-Queen hypothesis29), and another that

asserts that microbial or phage lineages are stable, though very numerous convey-ing, like in a zoetrope, an illusion of a fast-paced evolutionary scenario. (A zoetrope is a 19th century device that produces an effect of apparent motion by rapidly displaying static pictures in succession).

Actually the question is much more relevant than just what is the time frame for change in the prokaryotic world. Some of us have proposed a Constant-Diversity (C-D) model30 in which we posit that there is a high diversity of concurrent clonal lineages within a single bacterial or archaeal population (within the same species). This diversity optimizes resource utilization by increasing the range of eco-logical niches that can be exploited. For example, in heterotrophic bacteria dif-ferent concurrent clones carry different sets of membrane transporters, increas-ing the range of organic compounds that the population can utilize efficiently. The result is a better exploitation of resources

Figure 1. (A) All-vs-all genomic comparison of several reference tailed bacteriophage genomes (non-labeled branches) and the 208 complete phage genomes retrieved in mizuno et al., 2013 (represented as squares at the end of the branches). clustering is based on a sequence similarity derived metric. the new genomic groups are labeled as G1 to G21. cGr: complete Genomic representative, sequences that had been assumed to represent complete phage genomes (mizuno et al., 2013). cGF: complete Genomic Fragment, sequences that from the comparison with the cGrs are known to be incom-plete phage genomes. (B) network view of similarity relationships within the novel phage groups. each phage contig (complete genome or incomplete) is represented by a node and edges between any two nodes indicate a high sequence identity (> 95%) and coverage of at least 20% of the shorter con-tig. the groups are boxed by the predicted host. the numbers of some of the genomic groups (G1-G21) to which the cluster of contigs belong are also indicated. Only clusters with more than two members are shown, 235 singletons and 47 doublets were also found but are not depicted in the figure.

e28265-4 Bacteriophage Volume 4

by a smaller number of different species. However, since prokaryotes reproduce clonally, that is, producing exact copies of themselves, there is the risk that one clone could be more successful (for example by acquiring an unusually increased share of the resources, or because of a tran-sient expansion of its niche). Such clonal sweeps are a known phenomenon in labo-ratory cultures in which a clone normally predominates in the long-term.31 Here is when phages come to the rescue. Since phage infection rate is density dependent, the inordinate increase of a clone would immediately make it more efficiently pre-dated upon by the populations of specific phages adapted to its receptors, following a classical Lotka-Volterra predator/prey equilibrium. This idea has repercussions at multiple levels, from biotechnology to food microbiology or human health (through the microbiome).

This model can be taken beyond the purely mechanistic level since it implies that the bacterial population actually includes its associated phages as a sort of tax collectors that equate and pre-serve the efficiency of the system mak-ing this consortium of cell clones, plus the phages preying on them, a sort of meta-clonal organism that can be selected upon or extinguished i.e., a unit of selec-tion or selectome.28 Our results support this view, as the local diversity found at a single marine water column sample is astonishing. Even within relatively simi-lar genomes abundant microdiversity could be established and still some hyper-diversified regions are found identical in genomes isolated from distant locations including the Atlantic and Pacific Oceans.

However, some results appear puzzling from a C-D perspective. For example, most phage genomes recruit unevenly from a

metavirome from the same location,32 as is the case of prokaryotic genomes, and as the C-D model would predict.30,33 On the other hand, some groups (specifically putative pelagiphages) recruited much more evenly and had no equivalent of metagenomic islands. It is possible that these are generalistic phages that do not have hyper-diversified recognition mod-ules.34 The under-recruiting genes were often involved in host recognition, for example the tail fiber genes, as predicted by C-D. But other under-recruiting genes, such as some terminases, appeared totally disconnected from host recognition.

Maybe we are underestimating the complexity of phage behavior. For exam-ple, different strategies of phage survival have been proposed in which some are slow growers with small burst size while others produce large burst sizes.35 Some are specialized in infecting a very narrow

Figure 2. (A) Patterns in local diversity: Schematic representation of four types of patterns seen in concurrent phage genomes (i) syntenic, nearly identical contigs with small insertions/deletions (ii) syntenic, nearly identical contigs with smaller, more divergent regions and (iii) flexible genomic islands in highly related contigs, (iv) typical mosaic architecture displayed by multiple, related contigs. (B) Global distribution of nearly identical phage fragments: On the left a map of the world is shown where three locations are marked in different colors, from left to right, South california coast, (Pacific Ocean, yellow), Bermuda (north Atlantic, orange) and the mediterranean (blue). On the right, genome fragments originating from different sources are compared (nucleotide sequence identity is shown). the filled circles next to the contigs indicate the isolation location (as shown in the map). the names of both genomic fragments originated from the mediterranean are abbreviated, e.g., S32-c64 for uvmed-cGr-c28-meddcm-Oct-S32-c64. length of the fragments and are also indicated.

www.landesbioscience.com Bacteriophage e28265-5

range of host while others have a wider range with the risk of failed infection or inferior productive yield.34,36 It is certainly possible that some genes that are diverse within the population are involved in the different intracellular environments of the host strains at multiple levels. Different terminases might help packaging differ-ent types of concatamers or with differ-ent types of capsids. The in-depth study of such hyper-diverse genes might help in understanding phage-host interactions at these different levels.

A Million Phage Genomes

With the advent of next-generation sequencing, we have already witnessed an exponential increase in the number of

microbial genomes that are being sequenced. However, phage genomics has remained largely untouched by this sequencing revo-lution. Given the advances in sequencing that are now unfolding, it is not unreason-able to finally tap the largest genetic reser-voir on the planet. The methods described here, in combination with new methods of single-molecule sequencing actually provide finally an uncomplicated way to interrogate complete communities and their associated phages. It is only a matter of time before we will be on the threshold of being able to track evolutionary processes in real-time in natural habitats.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by projects MAGYK (BIO2008-02444), MICROGEN (Programa CONSOLIDER-INGENIO 2010 CDS2009-00006), CGL2009-12651-C02-01 from the Spanish Ministerio de Ciencia e Innovación, DIMEGEN (PROMETEO/2010/089) and ACOMP/2009/155 from the Generalitat Valenciana and MaCuMBA Project 311975 of the European Commission FP7. FEDER funds sup-ported this project. RG was supported by a Juan de la Cierva scholarship from the Spanish Ministerio de Ciencia e Innovación.

References1. Tringe SG, von Mering C, Kobayashi A, Salamov AA,

Chen K, Chang HW, Podar M, Short JM, Mathur EJ, Detter JC, et al. Comparative metagenomics of microbial communities. Science 2005; 308:554-7; PMID:15845853; http://dx.doi.org/10.1126/science.1107851

2. Tettelin H, Masignani V, Cieslewicz MJ, Donati C, Medini D, Ward NL, Angiuoli SV, Crabtree J, Jones AL, Durkin AS, et al. Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: impli-cations for the microbial “pan-genome”. Proc Natl Acad Sci U S A 2005; 102:13950-5; PMID:16172379; http://dx.doi.org/10.1073/pnas.0506758102

3. Read BA, Kegel J, Klute MJ, Kuo A, Lefebvre SC, Maumus F, Mayer C, Miller J, Monier A, Salamov A, et al.; Emiliania huxleyi Annotation Consortium. Pan genome of the phytoplankton Emiliania under-pins its global distribution. Nature 2013; 499:209-13; PMID:23760476; http://dx.doi.org/10.1038/nature12221

4. DeLong EF. Preface. Microbial metagenomics, metatranscriptomics, and metaproteomics. Methods Enzymol 2013; 531:xxi; PMID:24060136; http://dx.doi.org/10.1016/B978-0-12-407863-5.09983-4

5. Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, Wu D, Paulsen I, Nelson KE, Nelson W, et al. Environmental genome shotgun sequencing of the Sargasso Sea. Science 2004; 304:66-74; PMID:15001713; http://dx.doi.org/10.1126/science.1093857

6. Ghai R, Martin-Cuadrado AB, Molto AG, Heredia IG, Cabrera R, Martin J, Verdú M, Deschamps P, Moreira D, López-García P, et al. Metagenome of the Mediterranean deep chlorophyll maximum studied by direct and fosmid library 454 pyrosequencing. ISME J 2010; 4:1154-66; PMID:20393571; http://dx.doi.org/10.1038/ismej.2010.44

7. Ghai R, Rodriguez-Valera F, McMahon KD, Toyama D, Rinke R, Cristina Souza de Oliveira T, Wagner Garcia J, Pellon de Miranda F, Henrique-Silva F. Metagenomics of the water column in the pris-tine upper course of the Amazon river. PLoS One 2011; 6:e23785; PMID:21915244; http://dx.doi.org/10.1371/journal.pone.0023785

8. Proctor LM, Okubo A, Fuhrman JA. Calibrating estimates of phage-induced mortality in marine bac-teria: Ultrastructural studies of marine bacteriophage development from one-step growth experiments. Microb Ecol 1993; 25:161-82; PMID:24189813; http://dx.doi.org/10.1007/BF00177193

9. Suttle CA. Marine viruses--major players in the global ecosystem. Nat Rev Microbiol 2007; 5:801-12; PMID:17853907; http://dx.doi.org/10.1038/nrmicro1750

10. Rohwer F, Edwards R. The Phage Proteomic Tree: a genome-based taxonomy for phage. J Bacteriol 2002; 184:4529-35; PMID:12142423; http://dx.doi.org/10.1128/JB.184.16.4529-4535.2002

11. Paul JH, Sullivan MB. Marine phage genom-ics: what have we learned? Curr Opin Biotechnol 2005; 16:299-307; PMID:15961031; http://dx.doi.org/10.1016/j.copbio.2005.03.007

12. Casas V, Rohwer F. Phage metagenomics. Methods Enzymol 2007; 421:259-68; PMID:17352928; http://dx.doi.org/10.1016/S0076-6879(06)21020-6

13. Angly FE, Felts B, Breitbart M, Salamon P, Edwards RA, Carlson C, Chan AM, Haynes M, Kelley S, Liu H, et al. The marine viromes of four oceanic regions. PLoS Biol 2006; 4:e368; PMID:17090214; http://dx.doi.org/10.1371/journal.pbio.0040368

14. DeLong EF, Preston CM, Mincer T, Rich V, Hallam SJ, Frigaard NU, Martinez A, Sullivan MB, Edwards R, Brito BR, et al. Community genomics among stratified microbial assemblages in the ocean’s inte-rior. Science 2006; 311:496-503; PMID:16439655; http://dx.doi.org/10.1126/science.1120250

15. Martín-Cuadrado AB, López-García P, Alba JC, Moreira D, Monticelli L, Strittmatter A, Gottschalk G, Rodríguez-Valera F. Metagenomics of the deep Mediterranean, a warm bathypelagic habitat. PLoS One 2007; 2:e914; PMID:17878949; http://dx.doi.org/10.1371/journal.pone.0000914

16. Mizuno CM, Rodriguez-Valera F, Garcia-Heredia I, Martin-Cuadrado AB, Ghai R. Reconstruction of novel cyanobacterial siphovirus genomes from Mediterranean metagenomic fosmids. Appl Environ Microbiol 2013; 79:688-95; PMID:23160125; http://dx.doi.org/10.1128/AEM.02742-12

17. Maniloff J, Ackermann HW. Taxonomy of bacte-rial viruses: establishment of tailed virus genera and the order Caudovirales. Arch Virol 1998; 143:2051-63; PMID:9856093; http://dx.doi.org/10.1007/s007050050442

18. Garcia-Heredia I, Martin-Cuadrado AB, Mojica FJ, Santos F, Mira A, Antón J, Rodriguez-Valera F. Reconstructing viral genomes from the environment using fosmid clones: the case of haloviruses. PLoS One 2012; 7:e33802; PMID:22479446; http://dx.doi.org/10.1371/journal.pone.0033802

19. Mizuno CM, Rodriguez-Valera F, Kimes NE, Ghai R. Expanding the marine virosphere using metagenomics. PLoS Genet 2013; 9:e1003987; PMID:24348267; http://dx.doi.org/10.1371/jour-nal.pgen.1003987

20. Freitas S, Hatosy S, Fuhrman JA, Huse SM, Welch DBM, Sogin ML, Martiny AC. Global distribution and diversity of marine Verrucomicrobia. ISME J 2012; 6:1499-505; PMID:22318305; http://dx.doi.org/10.1038/ismej.2012.3

21. Ghai R, Mizuno CM, Picazo A, Camacho A, Rodriguez-Valera F. Metagenomics uncov-ers a new group of low GC and ultra-small marine Actinobacteria. Sci Rep 2013; 3:2471; PMID:23959135; http://dx.doi.org/10.1038/srep02471

22. Angly F, Youle M, Nosrat B, Srinagesh S, Rodriguez-Brito B, McNairnie P, Deyanat-Yazdi G, Breitbart M, Rohwer F. Genomic analysis of multiple Roseophage SIO1 strains. Environ Microbiol 2009; 11:2863-73; PMID:19659499; http://dx.doi.org/10.1111/j.1462-2920.2009.02021.x

23. Garcia-Heredia I, Rodriguez-Valera F, Martin-Cuadrado AB. Novel group of podovirus infect-ing the marine bacterium Alteromonas macleodii. Bacteriophage 2013; 3:e24766; PMID:24228219; http://dx.doi.org/10.4161/bact.24766

24. Swan BK, Tupper B, Sczyrba A, Lauro FM, Martinez-Garcia M, González JM, Luo H, Wright JJ, Landry ZC, Hanson NW, et al. Prevalent genome streamlin-ing and latitudinal divergence of planktonic bacte-ria in the surface ocean. Proc Natl Acad Sci U S A 2013; 110:11463-8; PMID:23801761; http://dx.doi.org/10.1073/pnas.1304246110

25. López-Pérez M, Gonzaga A, Rodriguez-Valera F. Genomic diversity of “deep ecotype” Alteromonas macleodii isolates: evidence for Pan-Mediterranean clonal frames. Genome Biol Evol 2013; 5:1220-32; PMID:23729633; http://dx.doi.org/10.1093/gbe/evt089

e28265-6 Bacteriophage Volume 4

26. Zhao Y, Temperton B, Thrash JC, Schwalbach MS, Vergin KL, Landry ZC, Ellisman M, Deerinck T, Sullivan MB, Giovannoni SJ. Abundant SAR11 viruses in the ocean. Nature 2013; 494:357-60; PMID:23407494; http://dx.doi.org/10.1038/nature11921

27. Bollmann M, Bosch T, Colijn F, Ebinghaus R, Froese R, Guessow K, Khalilian S, Krastel S, Koertzinger A, Lagenbuch M. World ocean review 2010: living with the oceans. 2010

28. Rodriguez-Valera F, Ussery DW. Is the pan-genome also a pan-selectome? F1000Res 2012; 1:16; PMID:24358823

29. Stern A, Sorek R. The phage-host arms race: shap-ing the evolution of microbes. Bioessays 2011; 33:43-51; PMID:20979102; http://dx.doi.org/10.1002/bies.201000071

30. Rodriguez-Valera F, Martin-Cuadrado AB, Rodriguez-Brito B, Pasić L, Thingstad TF, Rohwer F, Mira A. Explaining microbial population genom-ics through phage predation. Nat Rev Microbiol 2009; 7:828-36; PMID:19834481; http://dx.doi.org/10.1038/nrmicro2235

31. Atwood KC, Schneider LK, Ryan FJ. Periodic selec-tion in Escherichia coli. Proc Natl Acad Sci U S A 1951; 37:146-55; PMID:14808170; http://dx.doi.org/10.1073/pnas.37.3.146

32. Mizuno CM, Ghai R, Rodriguez-Valera F. Evidence for Metaviromic Islands in Marine phages. Frontiers in Microbiology 2014; 5

33. Pasić L, Rodriguez-Mueller B, Martin-Cuadrado AB, Mira A, Rohwer F, Rodriguez-Valera F. Metagenomic islands of hyperhalophiles: the case of Salinibacter ruber. BMC Genomics 2009; 10:570; PMID:19951421; http://dx.doi.org/10.1186/1471-2164-10-570

34. Flores CO, Meyer JR, Valverde S, Farr L, Weitz JS. Statistical structure of host-phage interac-tions. Proc Natl Acad Sci U S A 2011; 108:E288-97; PMID:21709225; http://dx.doi.org/10.1073/pnas.1101595108

35. Koskella B, Meaden S. Understanding bacteriophage specificity in natural microbial communities. Viruses 2013; 5:806-23; PMID:23478639; http://dx.doi.org/10.3390/v5030806

36. Flores CO, Valverde S, Weitz JS. Multi-scale struc-ture and geographic drivers of cross-infection within marine bacteria and phages. ISME J 2013; 7:520-32; PMID:23178671; http://dx.doi.org/10.1038/ismej.2012.135